The importance of calcium (Ca2+) as a cell regulator is well established in eukaryotes. However, the role of Ca2+ in prokaryotes still remains elusive. Recent experimental work has suggested that Ca2+ ions may play a regulatory role in prokaryotic organisms. An essential step toward an increased understanding of the role of Ca2+ is the identification and characterization of Ca2+ binding proteins (CaBPs). In eukaryotes, CaBPs are involved in the regulation of multiple cellular events including: cell differentiation, gene expression, transport mechanisms and others. Our preliminary data indicates that several CaBPs are present in bacteria (E. coli, B. subtilis and B. pertussis). These proteins share similar biochemical characteristics with eukaryotic CaBPs, including calmodulin (CaM). In an effort to isolate and sequence these proteins we analyzed crude cell lysates by 2D-electrophoresis followed by mass spectrometry. Most of the proteins found with CaBP characteristics are associated with stress responses. Based on these initial findings and other published data the purpose of this application is: 1) to map changes in protein expression as a function of cytosolic Ca2+ levels during both optimal growth conditions and during induced stress responses (heat/cold/pH shock). 2) to study the role of Ca2+ ions in transcription and establish the nature of the calcium regulon. The long-term goal of this research is to illuminate the role of Ca2+ in bacteria. We hypothesize that CaBPs play an important role in Ca2+ homeostasis and that Ca2+ ions are involved in the regulation of several intracellular processes in bacterial cells. In particular, we hypothesize that the intracellular concentration of Ca2+ ions are involved in the regulation of stress response proteins with associated changes in gene expression. The following specific aims were developed: 1) To characterize further the previously identified CaBPs and to identify new CaBPs that are involved in the bacterial response to changes in calcium levels 2) To identify and characterize the adaptive mechanisms utilized by cells to maintain calcium homeostasis 3) To determine the role of calcium in stress-related proteomic changes 4) To investigate the specific genomic changes that occur in bacteria in response to changing calcium levels. This study will lead to better understanding of basic cell biology and physiology of both pathogenic and environmental microorganisms and may lead to development of new therapeutics for the control of pathogenic bacteria.